Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1
Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1
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DOI：
10.1038/sj.emboj.7600701
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发表时间：
2005-07-06
期刊：
影响因子：
11.4
通讯作者：
Ficner, R
中科院分区：
文献类型：
--
作者：
Strasser, A;Dickmanns, A;Ficner, R
在高等真核生物中，剪接体UsnRNPs的生物发生涉及核细胞质穿梭循环。在m(7) g帽依赖性snrna U1， U2， U4和U5输出到细胞质后，这些snrna中的每一个都与7个Sm蛋白相关。随后，m(7)G-帽被超甲基化成2,2,7-三甲基鸟苷(m(3)G)-帽。进口适配器snurportin1识别m(3)G-cap，并通过绑定importin-beta促进UsnRNPs的核进口。在这里，我们报道了snurportin1与结合的m(3)GpppG的m(3) g -cap结合域在2.4埃分辨率下的晶体结构，揭示了与mrna - guanlytransferase的结构相似性。Snurportin1结合高甲基化帽和RNA的第一个核苷酸，形成堆叠构象。这种结合方式与m(7) g -cap结合蛋白Cap-binding protein 20 （CBP20）、真核起始因子4E （eIF4E）和病毒蛋白39 （VP39）的结合方式明显不同。荧光光谱法评估了snurportin1对m(3)G-cap识别的特异性，证明了高度溶剂暴露色氨酸对m(7)G-cap rna识别的重要性。这种色氨酸以及另一种色氨酸在RNA碱基序列中的关键作用已通过核输入试验和几种snurportin1突变体的帽结合活性试验得到证实。
In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m(7) G-cap-dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m(7)G-cap is hypermethylated to the 2,2,7-trimethylguanosine (m(3)G)-cap. The import adaptor snurportin1 recognises the m(3)G-cap and facilitates the nuclear import of the UsnRNPs by binding to importin-beta. Here we report the crystal structure of the m(3)G-cap-binding domain of snurportin1 with bound m(3)GpppG at 2.4 angstrom resolution, revealing a structural similarity to the mRNA-guanylytransferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m(7)G-cap-binding proteins Cap-binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m(3)G-cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m(7)G-capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap-binding activity tests using several snurportin1 mutants.